Methods for detecting the presence of expanded cgg repeats in the fmr1 gene 5&#39; untranslated region

ABSTRACT

The invention provides improved methods for detecting the presence of expanded CGG repeats in the fragile X mental retardation 1 (FMR1) gene and for quantifying the amount of protein produced by the gene.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/799,873, which claims the benefit of U.S. Provisional Application60/832,024, filed Jul. 19, 2006, the contents of which are herebyincorporated by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made with government support under Grant No.AG024488, awarded by the National Institutes on Aging, and Grant No.HD40661, awarded by the National Institute of Child Health and HumanDevelopment. The government has certain rights in the invention.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

NOT APPLICABLE

BACKGROUND OF THE INVENTION

Fragile X syndrome (“FrX” or “FXS”) is the most common inherited form ofmental retardation in males, with reported incidences of 1 in 4000 inmales and 1 in 8000 in females. FXS is caused by the absence or areduced level of the protein encoded by the fragile X mental retardation(“FMR1”) gene; the gene is generally turned off when a CGG DNA repeatwith a non-coding portion of the gene is expanded to greater than 200CGG repeats. The FMR1 gene is on the X chromosome, and is located atXq27.3. The gene has a length of 38 kb and encodes a 4.4 kb transcriptwith 17 exons. (O'Donnell and Warren, Annu Rev Neurosci 25:31538 (2002).The gene sequence is publicly available under accession number L29074 inthe National Center for Biotechnology Information (NCBI) “EntrezNucleotide” database via the NCBI website. The fact that females havetwo alleles for the gene (one on each X chromosome) while males haveonly one (since they carry one X chromosome and one Y chromosome)accounts for the difference in incidence and severity between the maleand female populations.

Fragile X syndrome results from the presence of too many copies of thetrinucleotide CGG repeat in the 5′ untranslated region (“UTR”) of theFMR1 gene. Most people carry between about 6 and 40 trinucleotiderepeats. In persons with over 200 trinucleotide repeats (known as a“full mutation”), the repeats are generally hypermethylated, and thegene is silenced.

Persons are considered to have a premutation expansion of the FMR1 geneif they have between about 55 to 200 trinucleotide repeats, and to havea full mutation when they have more than 200 repeats. Males and somefemales with either premutation or full mutation forms of the gene(alleles) are considered to be carriers. Individuals with permutationalleles are at increased risk for developing fragile X-related disordersas adults. Approximately 1 in 5 adult women premutation carriers willexperience premature ovarian failure (“POF”) and approximately 40% ofmen over 50 years of age, and a smaller number of women, who areidentified through known fragile x families, will develop theneurodegenerative disorder, fragile X-associated tremor/ataxia syndrome(FXTAS) (Jacquemont et al., Lancet Neurol 6:45 (2007)). Both males andfemales with full mutation forms of the FMR1 gene generally developfeatures of the child-onset disorder, FXS, although females are usuallyless affected than are men since they typically have a second allele ofthe gene without the mutation, which provides some expression of theprotein encoded by the FMR1 gene.

Better identification of carriers and earlier identification of infantswith Fragile X syndrome could be accomplished by screening of both thegeneral population and of newborns, in particular, for expanded allelesof the gene. (Bailey, D. B. Jr. et al., Ment Retard Dev Disabil Res Rev10:3-10 (2004)). Early intervention in infants and toddlers withdevelopmental delay (intellectual disability), and associated behavioralproblems (e.g., autism), focusing on language, motor, social andcognitive development, results in improved developmental and behavioraloutcomes. (See, e.g., Guralnick, M. J. Am J Ment Retard 102:319-45(1998); Shonkoff, J. P. et al., Handbook of Early ChildhoodIntervention. New York: Cambridge University Press, (2000); Bailey, D.B. Jr. et al., Ment Retard Dev Disabil Res Rev 10:3-10 (2004)).

No prospective, controlled studies have been carried out, however, thatspecifically examine the efficacy of early intervention in fragile Xsyndrome. Early diagnosis would not only permit the study ofintervention in such cases, but would allow families to obtain geneticcounseling at a time that will make a difference for subsequentpregnancies (Bailey, D. B. Jr. et al., Ment Retard Dev Disabil Res Rev10:3-10 (2004)). In addition, early diagnosis will become even moreimportant as newer psychopharmacological treatments are developedspecifically for FXS (e.g., mGluR5 receptor antagonists; Hagerman, R. J.“Fragile X Syndrome: Diagnosis, Treatment and Research” Baltimore: TheJohns Hopkins University Press” 287-338 (2002); Berry-Kravis, E. et al.,Ment Retard Dev Disabil Res Rev 10:42-8 (2004)).

The need for detecting carriers of premutation alleles is increased byrecent findings that premutation alleles in females have been found tobe associated with premature ovarian failure (Allingham-Hawkins, D. J.et al., Am J Med Genet 83:322-5 (1999); Sullivan, A. K. et al., HumReprod 20: 402-12. Epub 2004 (Dec. 17, 2005)). Moreover, a second formof clinical involvement has recently been identified among older malecarriers of premutation (FMR1) alleles (Hagerman, R. J. et al., X.Neurology 57:127-30 (2001)), consisting of progressive intention tremor,gait ataxia, Parkinsonism, and autonomic dysfunction; this disorder hasbeen designated “fragile X-associated tremor/ataxia syndrome” (FXTAS).An effective screening tool would reduce the number of missed orincorrect diagnoses for both POF and FXTAS.

It is estimated that at least one-third of all adult male premutationcarriers over 50 years of age, who are ascertained through known fragileX families, will develop symptoms of FXTAS, and the penetrance appearsto increase with age (Jacquemont, S. et al., JAMA, 291:460-69 (2004)).Given the carrier frequency among males of ˜1/800 (Dombrowski, C. etal., Hum Mol Genet 11:371-8 (2002)), FXTAS appears to be one of the morecommon single-gene forms of tremor and ataxia among older adult males inthe general population.

An effective screening tool for expanded alleles of the FMR1 gene mustsatisfy several tests: it must be able to reliably detect and sizeexpanded alleles at least through the upper portion of the premutationrange; it must be rapid in both primary detection and secondary analysisphases and identify all alleles in the full mutation range for bothmales and females; it must be able to unambiguously distinguish betweenfemales who are homozygous for normal FMR1 alleles (single normal bandfollowing polymerase chain reaction (“PCR”); ˜40% of all females) andfemales with one normal allele and a second, full mutation allele thatdoes not PCR amplify (single normal band, apparent homozygote); thisthird test has been the greatest impediment to high-throughputscreening. Finally, the test should be inexpensive enough for largescale screening.

A number of approaches were considered in trying to develop a screeningtool that meets the tests noted above. Strategies considered includedfluor-labeled, PCR-based fragment analysis, and Long PCR methods basedon bisulfite modification. For example, (“automated”) fluorescentprobe-based fragment analysis currently fails all the tests. The methodcannot reliably detect premutation alleles throughout the premutationrange, particularly in females, due to the rapidly diminishing signalstrength of the expanded allele with increasing CGG repeat number; thislatter issue requires significant operator involvement for theinterpretation of each scan, thus dramatically reducing the throughputof the method. Moreover, the method is too expensive, due to the costsassociated with capillary matrix, fluorescent reagents (e.g., primers),and instrument service and overhead.

Genotyping analysis is estimated to cost $15-20 per sample on a thousandsample basis. The method does not provide consistent, positive readsthroughout the premutation range, particularly for DNA samples fromfemale carriers. Furthermore, the method requires substantial time foroperation and interpretation. Finally, the method does not reliablydistinguish between normal homozygous females and the presence of a verylarge (non-PCR amplifiable) full mutation allele. Therefore, while thefragment analysis approach holds great potential for rapid, automatedscreening, the technology is not currently sufficiently developed to beused as a screening/testing tool.

PCR approaches based on bisulfite modification of the CGG repeatsequence (conversion of the unmethylated C nucleotides to U nucleotides)(Clark, S. J. et al., Nucleic Acids Res 22:2990-7 (1994)) hold promisefor subsequent long PCR amplification, since the bisulfite-modified DNAis both lower in CG-content and of lower sequence symmetry (base pairchanges within the CGG repeat element). However, the bisulfite treatmentdoes not reliably preserve sufficient DNA for PCR from small DNAsamples, such as those obtained from blood spots, due to the well-knowndegradation of DNA during the bisulfite conversion process (Grunau, C.et al., Nucleic Acids Res 29:E65-5 (2001)). Thus, whereas some samplescan be genotyped using this approach, the method can fail unpredictablydue to sample degradation during the bisulfite treatment. This methodalso requires additional steps in the screening process, including timeand additional steps for bisulfite conversion of DNA. Approximately 6hours are required for a complete conversion, somewhat less time if onlypartial conversion is required, which adds to the cost through operatortime. Finally, it is very important for a successful and reliableanalysis that high-quality DNA is used, which is the major pitfall ofthe bisulfate approach.

Thus, the ability to screen for persons with large numbers oftrinucleotide repeats in the FMR1 gene is of considerable importance.The present invention fills these and other needs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. FIG. 1A. Photo of a gel showing secondary screening methodby PCR to resolve apparent homozygosity in females. The approach uses acombination of betaine and a chimeric (CGG-targeted) PCR primer thatprimes randomly, but with a size bias for amplification of smallerexpansions, within the CGG repeat. FIG. 1B shows a schematic of thebinding of primers to produce the results shown in the lanes of the gel.The PCR of normal alleles using the art standard “c” and “f” primers isshown in lane 2 of the gel (FIG. 1A) and in the schematic labeled lane 2of FIG. 1B. PCR of normal alleles using the “c” primer (shown as an openarrow in FIG. 1B) and the chimeric primer (shown as a line arrow in FIG.1B) results in only small PCR products, as shown in lane 4 of the gel,FIG. 1A, and in the schematic labeled as lane 4 in FIG. 1B. In contrast,PCR of large alleles using the “c” primer and the chimeric primerresults in an extensive smear, as shown in lane 5 of the gel (FIG. 1A)and the schematic labeled “Lane 5” in FIG. 1B, reflecting priming withinthe extended CGG repeat. For full mutation alleles, priming by thestandard downstream primer (primer “f”) does not occur. Representationof the PCR products for each lane is given to the right of the gelimage. The chimeric primer comprises a 3′ (CCG)₄ (SEQ ID NO.:11) blockfor targeting and a 5′ random N24 block for subsequent amplification[N₂₄-(CGG)₄].

FIG. 2. FIG. 2 shows the 5′UTR immediately upstream of the start of thecoding region of the FMR1 gene, the start codon, and the beginning ofthe coding sequence (SEQ ID NO:15). The start codon, ATG is capitalizedand underlined. The CGG repeat region is in bold and underlined.Nucleotide position numbering is as set forth in the sequence set forthin the Entrez Nucleotide database under accession number L29074.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides long sought solutions to problems thathave impeded screening for and analysis of expanded repeats of CGG inthe 5′ untranslated region (“UTR”) of the FMR1 gene. These solutionspermit determining whether an individual either has a premutation in thegene (indicated by 55 to up to 200 repeats) and is therefore a carrier,or has 200 or more repeats, and therefore has a full mutation in thegene. In persons with a full mutation, the repeat region becomeshypermethylated, silencing the gene. The lack of the protein, FMRP,encoded by the gene adversely affects neurological development andresults in Fragile X Syndrome (“FXS”).

The solutions of the invention permit large scale, cost effectivescreening for infants and toddlers at risk of development of FXS and forwomen who are carriers of a large (e.g., full mutation) allele fromtheir normal, homozygous counterparts. Further, the solutions permitquantitating the amount of the FMRP actually expressed in individuals,thereby improving the ability to determine the degree to which aparticular individual may be affected by FXS. Since early interventionand treatment of such individuals is correlated with substantiallyimproved outcomes, the ability to diagnose early persons with FXS canreduce the burden of this disorder on the individual, on theindividual's family, and on society.

A. Improvement of PCR Assaying and Detecting Females with FullMutations.

In some embodiments, the invention provides improved methods ofidentifying premutation and full mutation alleles of up to ˜300 CGGrepeats by PCR, which can be used for high-throughput, low costscreening. Currently available techniques of PCR have not permittedidentification of persons with high numbers of repeats, especially infemales, and have not been adaptable for high-throughput screening. Themethods of the invention permit detection of numbers of repeats wellabove the upper end of the premutation range, and are applicable forboth males and females.

The methods can be used on small sample amounts, such as blood spots.This is an important advantage, since it permits the test to beintegrated, if desired, with the routine screening of newborns alreadyconducted in every state for phenylketonuria (PKU). PKU testing usuallyconsists of having the child's heel pricked and a few drops of bloodplaced on a card and sent to a lab for determination of bloodphenylalanine levels. The inventive methods allow the blood spots on thecard to also be used to determine whether a boy or girl has an expandedFMR1 allele, permitting routine and large scale screening for theseconditions.

Application of PCR to amplification of FMR1 genes with more than about100 to 150 repeats of CGG has been difficult because the high CG contenthas made separating the strands too difficult for standard techniques.This has permitted detection of only some of the persons withpermutation alleles. Further, because females have two FMR1 alleles, oneon each X chromosome, attempts to amplify the CGG-containing 5′untranslated region (UTR) in females with one normal allele and oneallele in which there are a high number of repeats has resulted in theamplification of the normal allele, concealing the presence of thehigh-repeat allele.

The methods of the invention provide a solution to both of theseproblems. First, the methods modify a commercially available system, theExpand Long Template PCR system (Roche Diagnostics, Mannheim, Germany)by using higher than normal concentrations of the osmolyte betaine(N,N,N-trimethylglycine). Frackman et al., Promega Notes 65:27 (1998),note that a variety of additives and agents can be included in PCRamplifications to increase yield, specificity and consistency. Theirlist of such agents includes dimethyl sulfoxide (DMSO), betaine,formamide, glycerol, nonionic detergents, bovine serum albumin,polyethylene glycol and tetramethylammonium chloride. DMSO is noted tobe useful in disrupting base pairing, whereas betaine is noted toequalize the contribution of G-C and A-T base pairing to the stabilityof the DNA duplex. Id. When used with high G-C content DNA, Frackman etal. recommend the use of 1 M betaine, while others recommend the use of1.3 M betaine with 1.3% DMSO. Studies underlying the present inventionfound that the use of betaine at 1.3M did not result in the ability todetect persons with 200 repeats.

Surprisingly, increasing the concentration of betaine from 1.7 M toabout 2.2 M permitted detecting CGG repeats at numbers ranging fromnormal (4-54) throughout the premutation range in both males andfemales. The PCR products could be directly visualized on agarose gelafter ethidium bromide staining; subsequently, the size of the allelescan be more precisely sized on acrylamide gels or by fragment analysis,if required.

While this by itself is a useful and important advance, it does not alsosolve the problem of how to provide low-cost, high-throughput screeningfor heterozygous females. One of skill will appreciate that, for males,one will see only one band upon PCR, since males only have one allele.Females, in contrast, should show two bands, one for each allele. Theproblem arises from the fact that, in 40% of females, only one band willbe visible. This can arise, however, from either of two very differentpossibilities, which are best explained by presenting three scenarios.In the first scenario, the female has two normal alleles, each of whichhaving approximately the same number of repeats. In this scenario, onlyone band will be visible on the gel due to indistinguishability of thetwo alleles. In the second scenario, the female has one allele with anormal number (e.g., 30) of repeats, and a premutation expansion (e.g.,150 repeats) for the second allele. In this scenario, two bands will bevisible, one (the allele with 30 repeats) being fairly dark, and thesecond one being light, but visible. In the third scenario, the femalehas one normal allele, with 30 repeats, and one allele with a fullmutation, with 400 repeats. In this scenario, only one band, for thenormal allele, will be visible. PCR will not amplify the second allele.

The problem is that the first scenario describes some 40% of females,while the third scenario describes one in 3000. Thus, out of 10,000females, 4000 will show a single band, of which 3 will have a fullmutation. PCR alone does not permit distinguishing between the 3997 withtwo normal alleles and the 3 with a full mutation of one allele. Thus,standard PCR, even with the improvement of increasing betaineconcentration does not permit rapid, high-throughput assaying forfemales with full mutations.

The present invention solves this problem. Surprisingly, it rests inpart on the realization that one does not have to amplify the entire CGGrepeat region to determine the presence of a full mutation. Rather, thepresent invention stems in part from the realization that a primaryscreening tool does not need to define the exact size of a full mutationallele, but only to signal its presence. The problem of resolving whichof the 40% of females who appear homozygous have a full mutation canthen be determined by defining the size of the full mutation allele withmore traditional methods (e.g., Southern gels). Previous attempts tosolve this problem have failed in part because of the difficulty ofamplifying the full CGG repeat region.

Persons of skill are aware that the 5′ UTR region of the FMR1 gene hasbeen defined and, except for the portion that comprises the CGG repeats,is uniform among individuals (as noted in the Background, the entireFMR1 gene has been sequenced and made publicly available). Thus, thepractice in the art is to amplify the 5′ UTR region containing the CGGrepeat region by using primers which hybridize within the 5′ UTR in thesections flanking the CGG repeat region. Two widely used sets of suchprimers are the “c” and “f” primers (Fu Y H et al. Cell 67:1047 (1991)):5′-agccccgcacttc caccaccagctcctcca-3′ (SEQ ID NO.:1) and5′-gctcagctccgtttcggtttcacttccggt-3′ (SEQ ID NO.:2), and the “1” and “3”primers employed by Brown et al., JAMA, 270:1569-75 (1993). The “1” and“3” primers are on the outsides of the regions flanking the CGG repeatregion and therefore capture a larger portion of DNA, while the “c” andf primers result in a smaller amplicon and greater resolution. Neitherof these sets of primers, however, can amplify the CGG repeats of a fullmutation.

In the present invention, samples from females that display a singleband in the normal range (that is, females who could be a homozygote orcould have a normal allele and a full mutation) in a first round ofscreening are resolved by a second round of screening. The first roundof screening can use conventional primers, such as those of Fu or Brown.Any pair of upstream/downstream primers bracketing the CGG repeat regioncan be used for the first round screening, and it is understood withinthe art that such primers are selected with respect to stability andspecificity using commonly known programs for determining primers.Typically, the upstream primer will be a nucleotide sequencecomplementary to a portion of the 5′ untranslated region of the FMR1gene that is not within the CGG repeat region. Since the Fu and Brownprimers are readily available and work well, however, it is convenientto use them for in the first round screening.

In the second round, as usual, two primers are used for the PCRreaction. As for the first round screening, one primer is a nucleotidesequence complementary to a portion of the 5′ untranslated region of theFMR1 gene that is not within the CGG repeat region. Conveniently, thefirst primer can be one of the standard primers used in PCR of the CGGregion, such as the “c” primer of Fu or the “1” primer of Brown. Thesequence of the 5′ UTR upstream (that is, 5′) of the CGG repeat isknown, however, and the person of skill can readily design any number ofadditional, alternative 5′ primers using standard techniques. Generalguidelines for designing efficient and specific primers are well knownin the art and are taught in, for example, Innis, et al., eds., PCRPROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, 1990 (Academic Press,San Diego Calif.), and Dieffenbach and Dveksler, PCR PRIMER A LABORATORYMANUAL, 2003 (Cold Spring Harbor Laboratory Press, Cold Spring HarborN.Y.). In some preferred embodiments, the 5′ primer is the “c” primer orthe “1” primer, with the c primer being more preferred.

In the sequence set forth for the FMR1 gene in the Entrez Nucleotidedatabase, under accession no. L29074, the ATG start codon, which definesthe downstream end of the non-coding 5′UTR, commences at position 13962.The CGG repeat region starts at position 13833 and ends at position13962, as shown in FIG. 2. Persons of skill will appreciate that thedeposited sequence is that of a person with a normal allele. The numberof repeats in this region is highly variable within the humanpopulation, and pre-mutation or full mutation alleles will have more CGGrepeats than shown in FIG. 2. The nucleotides on either side of the CGGrepeat region, however, will be the same. SEQ ID NO.:12 sets forth thesequence of the gene upstream of the start codon, as set forth underaccession number L29074, as well as the start codon (commencing with the“a” at position 13962) and a portion of the beginning of the codingsequence as set forth in FIG. 2. As noted, the CGG repeat regioncommences at position 13833. Therefore, the first primer is designed tohybridize to portions of SEQ ID NO.:12 that are 5′ of position 13833.

As persons of skill will appreciate, the 5′UTR of a gene is that portionof the gene that is the template of the untranslated part of the RNAproduced by the gene. With respect to the FMR1 gene, the 5′UTR isconsidered to commence at approximately nucleotide 13701. For amplifyinga portion of the gene, the location of the 5′UTR is relativelyunimportant since primers can be selected to amplify kilobases ofgenome. Better resolution of the area of interest, however, is achievedby amplifying smaller sequences, and it is thus preferable to select a5′ primer that hybridizes relatively close to the CGG repeat region.Thus, the primer is preferably selected to hybridize to a region 500 orfewer nucleotides 5′ of position 13833, more preferably 400 or fewernucleotides 5′ of position 13833, more preferably 300 or fewernucleotides 5′ of position 13833, still more preferably 200 or fewernucleotides 5′ of position 13833, even more preferably 150 or fewernucleotides 5′ of position 13833, and most preferably 100 or fewernucleotides 5′ of position 13833. Persons of skill will also be awarethat the primer is preferably selected to hybridize to a portion of thegenomic DNA that has 40-60% A or T nucleotides so that the annealing ofthe primer to the genomic DNA is not more stable than desirable forready separation of the strands during the amplification cycles. Suchprimers can include, for example, a 20-mer complementary to positions13391 to 13409 of SEQ ID NO.:12, and a 20-mer complementary to positions13661 to 13680 of SEQ ID NO.:12. The “c” and “1” target the regiondefined by the sequence lying between 13701 and 13833. The “f” and “2”primers target sequence that lies downstream of 13892 in SEQ ID NO.:12;such sequence can include UTR sequence but can also include codingsequence downstream of the ATG.

Unlike the case for the first round PCR, the second primer comprises CCGrepeats that “sit down” randomly within the CGG repeat region instead ofin the flanking region. The second primer may vary in the number of CCGrepeats that it possesses, with numbers between 3 and 9 being preferredfor ease of synthesis, 4-8 repeats being more preferred, 4, 5, 6 or 7repeats being still more preferred, 4, 5, or 6 repeats being morepreferred yet, and 4 or 5 repeats being especially preferred. In someembodiments, four repeats are particularly preferred. Rather thanproviding a single band indicative of a full mutation, which has beenthe goal of previous, but unsuccessful methods, when applied to a samplefrom a person having an allele with a full mutation, the inventivemethod results in a broad smear as the result of amplicons of multipledifferent lengths. The smear is indicative of the presence of a fullmutation. CGG repeats were used in initial studies and can be used;however, better results were obtained with CCG repeats as the secondprimer, and are preferred. Use of either CCG repeats or CGG repeats arepreferred to any combination of CGG and CCG repeats in the same primer.

The invention also provides a solution to an additional concern. Thesecond primer can simply be a series of CCG repeats (complementary tothe CGG repeats in the strand amplified by the “c”, “1”, or equivalentprimer) as described above. Because a CCG primer sits randomly in therepeat area, over multiple cycles of amplification, the amplicons tendto become smaller in size, since random priming within the CGGrepeat-containing amplicons will always produce shorter products for agiven round than the amplicons produced by the preceding round. While apure (CCG)_(n) second primer is useful, it would be preferable to have asecond primer that results in maintaining the size of the ampliconsafter multiple cycles of amplification.

The invention solves this problem as well. In preferred embodiments, thesecond primer is a “chimeric” primer, so-called because it includes botha 3′ portion that is composed of CCG repeats as described in thepreceding paragraph, contiguous to a 5′ end that has a nucleotidesequence that is random. After one or more rounds of amplication,however, the resulting amplicons contain a sequence complementary to the5′ end of the chimeric primer. This sequence on the amplicons isperfectly complementary to the sequence on the 5′ end of the chimericprimer and, without wishing to be bound by theory, the hybridization ofthese perfectly complementary sequences is energetically favored. Thus,after the first few rounds of amplification, the size distribution ofthe amplicons is “locked in.”

Within the constraints noted below, the sequence on the 5′ end of thechimeric primer can be a varied sequence of approximately 12-60nucleotides, with 12-30 being preferred and 21-27 being more preferred.A 24 nucleotide sequence is particularly preferred, and for conveniencemay be referred to herein as “N24”. In the first cycle of amplification,the chimeric primer “sits” on the CGG repeat region of the FMR1 gene byhybridization of the CCG repeats of the primer to the CGG repeats of the5′UTR of the gene. Preferably, regardless of the length of theparticular sequence chosen for the 5′ end of the chimeric primer, A or Tnucleotides constitute between about 30% to 70%, or more preferably40-60%, of the total number (for example, 10 A or T nucleotides out of24 total would constitute 42% AT of the 5′ end of the primer). Further,repeats of CCG are preferably avoided in the 5′ end of the chimericprimer to avoid having it compete with the 3′ end in sitting down in theCGG repeat region.

Surprisingly, the chimeric primers of the invention are effective,whereas a pure CCG primer with 10 CCG repeats was not effective. Withoutwishing to be bound by theory, it is believed that primers with 10 ormore repeats of CCG or CGG may form undesirable secondary structuresthat could interfere with amplification.

In a preferred embodiment, the second, chimeric primer is a “(CCG)₄”with a N24 5′ end, such as: 5′-AGC GTC TAC TGT CTC GGC ACT TGC CCG CCGCCG CCG-3′ (SEQ ID NO.:4), where the underlined portion will sit downrandomly within the CGG repeat region in the FMR15′UTR. Since theseembodiments of the second primer have a 24 nucleotide 5′ end, theseembodiments of the chimeric primer can also be referred to as“N24(CCG)₄”. Other examples of N24(CCG)₄ chimeric primers include 5′-GACCTG TAT TGG GTC ACG TCA GTC CCG CCG CCG CCG-3′ (SEQ ID NO.:5), 5′-AGCGCT ATC TCT TCC AGA GCT TTC CCG CCG CCG CCG-3′ (SEQ ID NO.:6), and5′-GCT CGC TAC TGC TTC CGG TAC CGT CCG CCG CCG CCG-3′ (SEQ ID NO.:7). Asdescribed above, any of these chimeric primers could be varied by, forexample, omitting the first three nucleotides of the 5′ end to form an“N21” chimeric primer, such as 5′-GTC TAC TGT CTC GGC ACT TGC CCG CCGCCG CCG-3′ (SEQ ID NO.:8), or by having three additional nucleotides, toform an “N27” chimeric primer, such as a different number of CCGrepeats, 5′-ATT GCT CGC TAC TGC TTC CGG TAC CGT CCG CCG CCG CCG-3′ (SEQID NO.:9), or by varying the number of CCG repeats, to provide forexample 3 (for example, 5′-AGC GTC TAC TGT CTC GGC ACT TGC CCG CCGCCG-3′ (SEQ ID NO.:10), or six (for example, 5′-AGC GTC TAC TGT CTC GGCACT TGC CCG CCG CCG CCG CCG CCG-3′ (SEQ ID NO.:11). The practitionerwill appreciate that numerous additional chimeric primers can bedesigned following the teachings set forth herein.

PCR using a first primer in the 5′ UTR region preceding the CGG repeatregion (such as the “c” or “1” primers) and a second primer of theinvention gives rise to a large smear on the analytical agarose oracrylamine gel (or any other suitable analytical sizing medium orcolumn), either manual or automated, that indicates the presence ofexpanded alleles. An example is shown in FIG. 1A, lane 5. Since thissecond screen is only conducted on samples from females who have had asingle band (which indicates that they do not have an allele withrepeats that would fall into the premutation range), the smear indicatesthat the male or female has a full mutation. A sample from an individualwithout an expanded allele will not create a large smear. In thismanner, normal alleles of the same or nearly the same size in femalescan be readily distinguished from carriers of expanded alleles.

While simple, the presence of the smear signals the presence of asecond, large allele, whereas the absence of the smear confirms truehomozygosity. This method is extremely rapid, and is amenable to use inautomated procedures that allow thousands of samples to be screened perday. To enhance its use in automated procedures, the method can furtherbe extended to incorporate the use of fluorescent primers and capillaryor gel-based automated scanning procedures. Alternatively, the primersmay optionally be labeled at their 5′ ends with, for example,radionuclides. Labeling is, however, not necessary for the practice ofthe invention.

The present invention modifies the basic PCR protocol to allow directreads of unmodified DNA samples, for both males and females, throughoutthe premutation range. Furthermore, the method allows direct analysis byethidium staining on gels, since no modified nucleotides are requiredfor PCR amplification through the expanded CGG repeat.

As noted, the PCR methods of the invention preferably use the modifiedamino acid betaine. Betaine permits PCR amplification of very GC-richsequences, due to the destabilizing properties of the amino acid (withrespect to DNA), as well as its stabilizing action on the polymerases(Baskaran, N. et al., Genome Res 6:633-8 (1996)). Furthermore, betaineappears to allow the PCR reaction to overcome low levels of contaminantsthat can co-purify with DNA (Weissensteiner, T. et al., Biotechniques21:1102-8 (1996)). This is particularly important in protocols thatutilize blood spots, where contaminants on the filters may interferewith PCR reactions. In studies underlying the invention, this approachhas allowed PCR reactions to be run directly off of blood spots on theircollection papers without any need to even perform adenaturation/separation step prior to PCR amplification.

Use of the betaine protocol of the invention has allowed consistentdetection of expanded alleles throughout the premutation range for bothmales and females. This means that as a screening tool, all males can betyped, with no requirement for a secondary screening tool. Further, forfemales, all premutation carriers can be typed without the need for asecondary screening tool; that is, apparent homozygous females (singleband in the normal size range) will either have two normal alleles, orwill have a single normal allele with one full mutation allele. Asecondary screening tool utilizes the hybrid CGG-based primer todistinguish between homozygous normal and full mutationheterozygotes/mosaic females as described.

The betaine protocol alone allows consistent detection of expandedalleles throughout the premutation range for both males and females.This means that as a screening tool, all males can be typed with norequirement for a secondary screening tool. For females, this toolidentifies all premutation carriers without the need for a secondaryscreening tool; that is, apparent homozygous females (single band in thenormal size range) will either have two normal alleles, or will have asingle normal allele with one full mutation allele. A secondaryscreening tool will utilize the hybrid CGG-based primer to distinguishbetween homozygous normal and full mutation heterozygotes/mosaic femalesas described herein.

Although the screening protocol discussed above utilizes a DNA-based PCRamplification method, it is recognized that RNA based amplificationmethods are also applicable, where suitable DNA primers can behybridized to either side of the CGG repeat, as with the embodimentdescribed above, but with extension of the primer to include promotersfor various RNA polymerases. It is known that at least some RNApolymerases can transcribe through the entire CGG repeat even for fullmutation alleles, since FMR1 mRNA is produced in individuals with fullmutation alleles, repeated rounds of RNA transcription/amplificationwill produce RNA species that reflect the sizes of the original CGGrepeat element. Amplification methods which can be used in the practiceof the invention include amplification methods utilizing a catalytic RNAto replicate nucleic acids, U.S. Pat. No. 4,786,600; amplificationsystems based on strand displacement, see Walker, et al., EP 0 497 272;and different transcription-based amplification methods. Among thelatter are those of Malek, WO 91/02818; Kacian, et al., U.S. Pat. No.5,399,491; Kacian, et al., EP 0 587 266; and McDonough, et al., EP 0 587298.

B. Immunoassays for Fragile X

There are currently available methods for detecting the presence offragile X mental retardation protein (“FMRP”) in peripheral white bloodcells. The first is an immunocytochemical staining test using acommercially available mammalian antibody from clone 1C3 directedagainst FMRP, in which 100 to 200 lymphocytes are identified followingantibody based staining and the number of FMRP-positive cells(identified by positive staining) are counted. This method is, at best,an indirect measure of protein level, since it only scores the fractionof cells where the protein is detectable, not the level of protein inthose cells. The presumption is that higher protein levels will yieldmore positive-staining cells. Although this trend generally holds, oncemost cells stain positive, no conclusions regarding protein levels canbe made (ceiling effect); further, for very low protein levels, usuallythe case in the full mutation range, few if any cells will stainpositive for FMRP (floor effect). The other disadvantage with thismethod is that it is time consuming. A second method, Western blotanalysis, is not generally useful for detecting FMRP in non-transformedlymphocytes, due to the low protein levels in those cells, although itcan be used to study transformed (lymphoblastoid) cells. A third methodis hair root analysis, in which 10-20 hairs are plucked from the scalpand then subjected to the same staining procedure. This procedure can beproblematic since children with FXS are often tactilely defensive andfind the contact to pluck the hair difficult to tolerate. The methodsare reviewed in, for example, Willemsen and Oostra, American Journal ofMedical Genetics, Seminars in Medical Genetics 97(3):183-188 (2001).Since the degree of mental impairment correlates with FMRP levels inperipheral blood lymphocytes, a quantitative test would be highly usefulfor both diagnosis and for prognosis.

For at least a decade, groups have been trying to develop anenzyme-linked immunosorbent assay (ELISA) that would permit quantitationof the amount of FMRP. Unfortunately, no ELISA has been forthcoming. Inpart, this arises because of the presence of two closely relatedautosomal homologs, Fragile X mental retardation autosomal homolog 1(“FXR1”) and 2 (“FXR2”), that also tend to be detected by mostantibodies. Further, because of the similarity between mouse and humanFMRP, mouse antibodies, such as 1C3, tend to be less robust in theirbinding affinities to FMRP than is desirable for ELISA assays. Ingeneral, it has not been possible to develop a mammalian antibody tohuman FMRP due to the high sequence similarity between all (non-human)mammalian forms of the protein.

The present invention has succeeded in providing an ELISA that can beused to quantitate FMRP. A sequence from the C-terminal portion of theFMRP protein was selected since it is different from the C-terminus ofthe FXR1 and FXR2 proteins in an attempt to generate antibodies thatcould differentiate FMRP from the two homologs. Further, a non-mammalianantibody is used as the capture antibody. This combination provedsuccessful in providing the ability to quantitate FMRP in ELISAimmunoassays.

Polyclonal chicken IgY antibodies were found effective in detectingFMRP. The antibodies can be used in any immunoassay in which detectionof FMRP is desired. A number of immunoassays in which the antibodies canbe used are known in the art. For example, the antibodies can be labeledwith enzymes, radioisotopes, or fluorescent compounds for use in enzymeimmunoassays (EIA), radioimmunoassays (RIA) or fluorescence assays, canbe used in assay techniques such as agglutination or turbidimetry, ordetected on Western Blots.

In a preferred form, the immunoassay is an ELISA. The chicken IgYanti-FMRP antibodies are preferably used as the capture antibody. SinceELISA procedures and variations are well known in the art, they will notbe described in detail herein. The following discussion sets forth theprocedure for a preferred embodiment.

In brief, peripheral blood mononuclear cells (PBMCs) are isolated usingFicoll-Paque® PURE (GE Healthcare) or Vacutainer® CPT™ tubes (BectonDickinson, Franklin Lakes, N.J.). If desired, the cells can be stored inliquid nitrogen until being assayed. The capture antibody, affinitypurified chicken IgY, is coated on a well of an ELISA plate. Typically,the well is blocked with a blocking agent, such as 2% casein inphosphate buffered saline (PBS) containing 0.05% polyoxyethylene (20)sorbitan monolaurate (PBS-T). The PBMC proteins are extracted, diluted,and added to the coated and blocked well. The proteins are incubated foran appropriate time at room temperature with rocking A standard amountof purified FMRP protein is preferably added to a duplicate well topermit quantitation of signal. The wells are then washed with PBS-T anda detecting antibody, such as mouse monoclonal anti-FMRP, withappropriate dilution (e.g., 1:10,000), is added and the mixture isincubated at room temperature for an appropriate time (e.g., 8 hours).The wells are washed with PBS-T, and a secondary antibody, such asdonkey anti-mouse antibody, conjugated to a label (e.g., horseradishperoxidase) and at an appropriate dilution (e.g., 1:2000) in blockingbuffer, is added to the wells and incubated. The wells are washed againand an appropriate substrate is added to detect the presence of thelabel.

EXAMPLES Example 1

During the course of clinical/molecular studies of fragile X syndromeand premutation carriers, more than one-thousand samples (DNA and/orfrozen PBMCs) have been archived spanning the normal, premutation, andfull mutation size ranges. A sub-set of these samples, representing 980fully characterized (PCR plus Southern) DNA samples, has the followingcharacteristics:

Allele⁽¹⁾ (# CGG repeats) # males # females Controls (<45) 222 82 Grayzone (45-54) 40 18 Premutation (55-200) 159 210  Full mutation (>200)117 57 Mosaics⁽²⁾ 44(P/F); 22 MM 6 MM Total 604 373  ⁽¹⁾For females, thelarger allele is within the specified range. ⁽²⁾P/F, premutation/fullmutation mosaic; MM, methylation mosaic.

For the female controls, 30 (52%) were apparent homozygotes (verified asnormal alleles by Southern gel analysis). These samples, plus anadditional 300 samples not yet fully characterized from both peripheralblood leucocytes and PBMCs, represent an important resource for thevalidation of the proposed screening methodology.

Example 2

Initial development of the PCR method of the invention utilized bloodsamples (not blood spots). Genomic DNA was isolated using the PuregeneDNA Blood kit (Gentra, Inc., Minneapolis, Minn.). PCRs were performedusing the “c” and “f” primers (5′-agccccgcacttccaccaccagctcctcca-3′ (SEQID NO.:1); 5′-gctcagctccgtttcggtttcacttccggt-3′ (SEQ ID NO.:2)(Fu, Y. H.et al., Cell 67:1047-58 (1991)) and performed using the Expand LongTemplate PCR System (Roche Diagnostics, Mannheim, Germany). Reactionmixtures included buffer 2 (Roche kit), 500 μM dNTPs, 0.33 pM of eachprimer and 100-500 ng of genomic DNA. The PCR buffer also included 2.0 Mbetaine (B0300, Sigma-Aldrich, St. Louis, Mo.); this concentration wasbased on a series of PCR optimization experiments using betaineconcentrations from 1.3 to 2.2 M. Previous reports recommended aconcentration of 1.3 M (Baskaran, N. et al., Genome Res 6:633-8 (1996)).We found the cited concentration to be too low for efficient expansionof the CGG element. The expected constant region of the PCR product was221 bp.

To establish the optimal amplification conditions, two male premutationcarriers, carrying expanded alleles of 90 and ˜200 CGG repeats, and acontrol male with an allele of 20 CGG repeats were tested. Differentconcentration (1.3M, 1.5M, 1.7M, 2.0M and 2.2 M) were also tested.Optimal results were obtained with a range of betaine concentrations of1.7-2.0 M.

Although alleles larger than 250 CGG repeats became progressivelyfainter, a PCR band was still visible in a male with ˜330 CGGs (˜1,300bp). No amplification product (“amplicon”) was detected for alleles >330CGG, well above the lower bound (200 CGG repeats) of the full mutationrange. It is noteworthy that carrier females with alleles of at least160 CGGs yielded PCR products, where both the normal and expandedalleles were clearly visible as two distinct bands. The larger alleleappeared proportionally weaker with increasing repeat length, but wasstill visible and discrete in the higher premutation range. Differentmolar ratios of 7-deaza-dGTP/dGTP (thought to destabilize secondarystructures usually formed by CG-rich DNA sequences), in combination withthe Expand Long Template PCR kit, was examined to see if this furtherimproved results. Results with 7-deaza-dGTP were not reproducible,probably due to the weak ethidium bromide staining of DNA synthesizedwith this base analog. The combination of 7-deaza-dGTP and betaine didnot improve amplification and, in several cases, led to a completeabsence of the PCR product. Failure to detect a second band for anyfemale with high premutation alleles (˜200 CGG repeats) would convertthat case to the apparent homozygote class, which would therefore besubject to the secondary screen using the hybrid CGG primer.

Example 3

For samples from males, the betaine-PCR method is capable of specifyingthe status (normal, gray-zone, premutation, full mutation) for allsamples. Furthermore, for all categories except full mutation, the sizeof the allele can be determined. For apparent full mutation males(absence of a band), a secondary analysis would be performed by Southerngel to rule in/out full mutation status and to determine the size of theallele (although the latter operation is not formally part of thescreen). Absence of a band would be due either to true full mutationalleles or by occasional failure of the PCR reaction. Confirmation of afull mutation allele in such cases could also be performed using thesecond phase of the PCR-based screening protocol, as used for femaleswith a single band. This secondary screening method would rule out anyPCR reaction failure, which could have resulted in the absence of a bandfor a male. Based on the expectation of one true full mutation alleleper ˜1/3,000-5,000 samples, one Southern gel would be needed per ˜50,000samples from males.

Example 4

For samples from females, the betaine-PCR method is capable ofspecifying the status of all premutation alleles based on theexpectation of two bands (homozygous premutation alleles have not beendescribed in the literature). Thus, the betaine-PCR screening procedureis capable of yielding a true estimate for prevalence of premutationcarriers without the need for any additional testing. Note that any casewith a single band in the premutation range would be treated as apparenthomozygosity, and would be subjected to secondary screening, which wouldalso rule in or out mosaicism due to the presence of premutation plusfull mutation alleles; such cases would not significantly affect theprevalence estimates. For samples from females that display a singleband in the normal range (apparent homozygosity), the samples would besubjected to a secondary, CGG-directed primer-based PCR screening.

Example 5

The major difficulty encountered when screening for expanded alleles ofthe FMR1 gene is the ambiguity associated with a single band (apparenthomozygosity) in females, which represent ˜40-50% of female cases. Innearly all cases (˜99.9%), the single band represents true homozygosity(i.e., two alleles differing by 0-2 repeats) However, the problem fromthe screening standpoint (standard PCR test) is that it is not possibleto deduce which of the 40-50% of female cases with a single band have anon-amplifying full mutation (or a very high premutation).

To eliminate this ambiguity, the present invention uses a novel,PCR-based approach that involves a secondary PCR screen (of the apparenthomozygous females) that combines the betaine PCR method with a chimericprimer that targets the CGG region itself. When used in combination withthe standard primer c (Fu et al., 1991), the secondary PCR reactionproduces an extended smear of amplified species only if an expandedallele is present (from the premutation to the full mutation range).Thus, the secondary screen returns a “yes or no” to the question of thepresence of an expanded allele. The nature of the full mutation (ormosaics, which would automatically be flagged) can be characterized morefully in subsequent studies or clinical workup through more traditionalmethods (e.g., Southern blot); the purpose of the screen is to flagthose cases.

This approach has been successfully performed using DNA collected from15 normal females and 15 full mutation females, and using DNA isolatedfrom blood spots from two normal females and two full mutation females.In all cases, PCR reactions consistently produce amplified products,which are visible as large smears on agarose gels, only in the fullmutation females. The results were validated and confirmed by Southernblot analysis. Thus, the combination of the two PCR approaches allowsdetecting expanded FMR1 alleles in both females and males, regardless ofthe size, by PCR on blood spots. The methodology is therefore suitablefor screening by collecting and using a small amount of DNA from bloodspots, which never previously been successfully demonstrated or appliedin screening fragile X samples, particularly for females. Two additionaladvantages of the inventive PCR approach are (i) that it lends itself toautomation, and (ii) that no DNA primer or other DNA labeling isrequired.

Example 6

Female samples with a single band are subjected to a second round ofscreening, which utilizes the chimeric PCR approach. As for all otherPCR reactions, the standard primer “c” is used; however, in place of thestandard primer “f”, the chimeric primer, 5′N24(CCG)n, is used. The N24portion of the primer is mixed-sequence DNA that is approximately 50%AT. PCR amplification using “c” and the chimeric primer gives rise to anextended smear on the gel for expanded alleles. No smear has beendetected in any case of a normal allele. The smear is generated by theamplification of expanded alleles even in the premutation range.However, premutation alleles can be separated by the betaine (primary)PCR screening method.

Example 7 FMRP ELISA Methods:

Lymphocytes were separated from heparinized whole blood usingFicoll-Paque™ PLUS (Cat. No. 17-1440-02, GE Healthcare Bio-SciencesCorp., Piscataway, N.J.) and washed two times with phosphate bufferedsaline (“PBS”, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄)containing Complete (Cat. No. 11836153001, Roche) protease inhibitors bycentrifugation at 1000×g. A pellet containing approximately 2×10⁶ cellswas resuspended in M-Per Mammalian Protein Extraction Reagent (No.78503, Pierce, Rockford, Ill.) with 150 mM NaCl, Protease InhibitorCocktail Set III, 10 μg/ml antipain and 10 μg/ml chymostatin, androtated at 4° C. for two hours. Extracted proteins were spun at 14,000×gto pellet any non-solubilized cell debris, protein concentration ofrecovered supernatant was quantified using a colorimetric bicinchoninicacid assay (#23227, Pierce).

Affinity purified chicken IgY against the peptide sequenceKDRNQKKEKPDSVD (SEQ ID NO.:3) was prepared by Ayes Lab, Inc (Tigard,OR). 100 ul (per well) of 2 μg/ml IgY diluted in PBS (137 mM NaCl, 2 mMKCl, 4.3 mM Na₂HPO₄ 1.4 mM KH₂PO₄) was incubated in Lumitrac 600(Greiner) 96-well plates for 24-48 hours at 4° C. Unbound IgY wasdiscarded, wells were blocked with 250 μl ELISA blocking buffer (2%hydrolyzed casein, 0.05% polyoxyethylene (20) sorbitan monolaurate, inPBS) at 20° C. for 12 hours. Wells were washed with 250 μl of PBS threetimes. Protein lysates and FMRP were diluted in PBS and added to wells,100 μl sample volume per well, and incubated at 20° C. overnight. Wellswere washed three times with PBS followed by three washes with PBS-T(PBS containing 0.05% polyoxyethylene (20) sorbitan monolaurate), 250 μlper well. Detecting antibody, mouse monoclonal anti-FMRP (MAB2160,Chemicon, Temecula, Calif.) was diluted to 1:10,000-1:20,000 in ELISAblocking buffer. 100 μl of detecting antibody was added to each well,and incubated for 8 hours at 20° C. Wells were washed with 250 μl perwell of PBS-T five times. 100 μl per well of HRP conjugated donkeyanti-mouse antibody (715-035-150, Jackson ImmunoResearch) diluted1:5,000 in blocking buffer was added and incubated overnight at 20° C.Wells were washed with 250 μl PBS-T five times. 100 μl Lumigen PS-Atto(#PSA-100, Lumigen, Inc., Southfield, MI) luminescent substrate wasadded to each well and incubated for 5 minutes at room temperature.Luminescence accumulated over 2.5 sec was read for each well with amicroplate luminometer (LMax, Molecular Devices, Sunnyvale, Calif.).

All publications and patent documents cited herein are incorporated byreference in their entirety for all purposes to the same extent as ifeach individual publication or patent document were individually denotedto be incorporated. Citation of various references in this document isnot an admission that any particular reference is considered to be“prior art” to the invention.

1-12. (canceled)
 13. A method of detecting the presence of fragile Xmental retardation protein (FMRP) in a biological sample, said methodcomprising an immunoassay using a non-mammalian antibody whichspecifically recognizes a peptide having the sequence of SEQ ID NO.:3,wherein detecting binding of said antibody to said FMRP in saidimmunoassay indicates the presence of said protein in said sample.
 14. Amethod of claim 13, wherein said immunoassay is an enzyme-linkedimmunosorbent assay (ELISA).
 15. A method of claim 13, wherein saidnon-mammalian antibody is a chicken IgY antibody.
 16. A method of claim14, wherein said non-mammalian antibody is the capture antibody of saidELISA.
 17. A method of claim 13, wherein said detecting is by detectingthe presence of a luminescent substrate.
 18. A method of claim 13,wherein said detection permits quantifying the amount of said FMRPpresent in said sample.